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• W2022840078 abstract "Correct positioning of neurons during embryonic development of the brain depends, among other processes, on the proper transmission of the reelin signal into the migrating cells via the interplay of its receptors with cytoplasmic signal transducers. Cellular components of this signaling pathway characterized to date are cell surface receptors for reelin like apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR), and cadherin-related neuronal receptors, and intracellular components like Disabled-1 and the nonreceptor tyrosine kinase Fyn, which bind to the intracellular domains of the ApoER2 and VLDL receptor or of cadherin-related neuronal receptors, respectively. Here we show that ApoER2, but not VLDLR, also binds the family of JNK-interacting proteins (JIPs), which act as molecular scaffolds for the JNK-signaling pathway. The ApoER2 binding domain on JIP-2 does not overlap with the binding sites for MLK3, MKK7, and JNK. These results suggest that ApoER2 is able to assemble a multiprotein complex containing Disabled-1 and JIPs, together with their binding partners, to the cell surface of neurons. This complex might participate in ApoER2-specific reelin signaling and thus would explain the different phenotype of mice lacking the ApoER2 from that of VLDLR-deficient mice. Correct positioning of neurons during embryonic development of the brain depends, among other processes, on the proper transmission of the reelin signal into the migrating cells via the interplay of its receptors with cytoplasmic signal transducers. Cellular components of this signaling pathway characterized to date are cell surface receptors for reelin like apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR), and cadherin-related neuronal receptors, and intracellular components like Disabled-1 and the nonreceptor tyrosine kinase Fyn, which bind to the intracellular domains of the ApoER2 and VLDL receptor or of cadherin-related neuronal receptors, respectively. Here we show that ApoER2, but not VLDLR, also binds the family of JNK-interacting proteins (JIPs), which act as molecular scaffolds for the JNK-signaling pathway. The ApoER2 binding domain on JIP-2 does not overlap with the binding sites for MLK3, MKK7, and JNK. These results suggest that ApoER2 is able to assemble a multiprotein complex containing Disabled-1 and JIPs, together with their binding partners, to the cell surface of neurons. This complex might participate in ApoER2-specific reelin signaling and thus would explain the different phenotype of mice lacking the ApoER2 from that of VLDLR-deficient mice. low density lipoprotein low density lipoprotein receptor very low density lipoprotein receptor Src homology 3 apolipoprotein E receptor 2 disabled-1 cadherin-related neuronal receptor c-Jun NH2-terminal kinase JNK interacting protein islet-brain-1 polymerase chain reaction hemagglutinin glutathione S-transferase protein interaction domain phosphate-buffered saline Members of the LDL1receptor (LDLR) family are endocytosis-competent cell surface receptors, which bind and internalize a wide variety of different ligands destined for removal from the circulation and the extracellular space, and/or serving as nutrients for certain cells. In accordance with this wide ligand spectrum and the numerous sites of expression in distinct cell types and organs, these receptors are involved in a variety of different biological processes (1Schneider W.J. Nimpf J. Bujo H. Curr. Opin. Lipidol. 1997; 8: 315-319Crossref PubMed Scopus (62) Google Scholar, 2Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1058) Google Scholar, 3Gliemann J. Biol. Chem. Hoppe-Seyler. 1998; 379: 951-964PubMed Google Scholar). The most recently discovered members of the receptor family are mammalian ApoER2 and its avian homologue LR7/8B (4Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Kim D.-H. Iijima H. Goto K. Sakai J. Ishii H. Kim H.-J. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Their common structure resembles that of the LDLR and the very low density lipoprotein receptor (VLDLR), but multiple alternative splicing events result in a complex array of receptor proteins (6Kim D.-H. Magoori K. Inoue T.R. Mao C.C. Kim H.-J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. J. Biol. Chem. 1997; 272: 8498-8504Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 7Brandes C. Novak S. Stockinger W. Herz J. Schneider W.J. Nimpf J. Genomics. 1997; 42: 185-191Crossref PubMed Scopus (50) Google Scholar). ApoER2 may contain a 59-amino acid insertion in its cytoplasmic domain, which is encoded by an extra exon that is differentially spliced in distinct variants of the receptor. The insertion is proline-rich and contains two potential SH3 binding sites (8Yu H. Chen J.K. Feng S. Dalgarno D.C. Brauer A.W. Schreiber S.L. Cell. 1994; 76: 933-945Abstract Full Text PDF PubMed Scopus (871) Google Scholar). The prevalent site of expression of ApoER2 and LR7/8B is the brain, particularly neurons and cells that are components of brain barrier systems, such as the epithelia of the choroid plexus and of the arachnoidea, as well as endothelial cells of blood vessels (9Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Clatworthy A.E. Stockinger W. Christie R.H. Schneider W.J. Nimpf J. Hyman B.T. Rebeck G.W. Neuroscience. 1999; 90: 903-911Crossref PubMed Scopus (54) Google Scholar). Recently, the finding that cytosolic adaptor proteins like Dab1 and Fe65 bind to the intracellular receptor domains, in particular to the NPXY motif of LDLR and the LDLR-related protein (11Trommsdorff M. Borg J.-P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33565Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 12Howell B.W. Lanier L.M. Frank R. Gertler F.B. Cooper J.A. Mol. Cell. Biol. 1999; 19: 5179-5188Crossref PubMed Scopus (333) Google Scholar), suggested that the current view of endocytosis of nutrients or removal of spent proteins as the main function of these receptors is only part of the story. Signal transduction should be considered to be another part of the functional spectrum that some or all of these receptors might participate in (13Willnow T.E. Nykjaer A. Herz J. Nat. Cell Biol. 1999; 1: E157-E162Crossref PubMed Scopus (185) Google Scholar). First proof for this hypothesis was generated by the results of targeted disruption of both the VLDLR and the ApoER2 in mice (14Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar). This genetic approach has identified both receptors as components of a signaling pathway that relays the reelin signal into migrating neurons. In addition, binding experiments showed that reelin interacts with the extracellular domains of both VLDLR and ApoER2 (15Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar, 16D'Arcangelo G. Homayoundi R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar). Reelin is secreted by Cajal-Retzius cells in the outermost layer of the developing cerebral cortex and orchestrates the migration of neurons along radial fibers, thus forming distinct cortical layers in the cerebrum (for review see Ref. 17Curran T. D'Arcangelo G. Brain. Res. Rev. 1998; 26: 285-294Crossref PubMed Scopus (214) Google Scholar). Similar mechanisms are used to establish the characteristic neuronal arrangement in the hippocampus as well as the correct positioning of Purkinje cells in the cerebellum. A naturally occurring genetic defect in reelin creates the phenotype of the reeler mouse, which is characterized by an inversion of the cortical layers and absence of cerebellar foliation with an abnormal distribution of the Purkinje cells. Naturally occurring disruption or targeted deletion of the DAB1 gene produces phenotypes indistinguishable from that of thereeler mouse, suggesting that Dab1 is another component in the reelin signaling pathway (18Sheldon M. Rice D.S. D'Arcangelo G. Yoneshima H. Nakajima K. Mikoshiba K. Howell B.W. Cooper J.A. Goldowitz D. Curran T. Nature. 1997; 389: 730-733Crossref PubMed Scopus (556) Google Scholar, 19Howell B.W. Hawkes R. Soriano P. Cooper J.A. Nature. 1997; 389: 733-737Crossref PubMed Scopus (618) Google Scholar). In addition, a family of cadherin-related cell surface proteins termed the cadherin-related neuronal receptor (CNR) family have been identified to bind reelin (20Senzaki K. Ogawa M. Yagi T. Cell. 1999; 99: 635-647Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). These proteins associate with Fyn, a nonreceptor tyrosine kinase of the Src kinase family (21Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1080) Google Scholar), which in turn interacts with Dab1 (22Howell B.W. Gertler F.B. Cooper J.A. EMBO J. 1997; 16: 121-132Crossref PubMed Scopus (301) Google Scholar). The current model suggests that reelin acts by direct binding to VLDLR and/or ApoER2 and to CNRs, thereby leading to phosphorylation of Dab1, which is associated with the tails of the LDLR relatives. Tyrosine phosphorylation of Dab1 may then start kinase cascade(s) controlling cellular motility and shape by acting on the neuronal cytoskeleton (14Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar,20Senzaki K. Ogawa M. Yagi T. Cell. 1999; 99: 635-647Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). However, other experimental evidence suggests that the Reelin/Dab1 signaling pathway might not be the only signaling event involving members of the LDLR family. Apolipoprotein E, a universal ligand for these receptors, exerts an antithrombotic activity by inhibiting platelet aggregation through the l-arginine:nitric oxide pathway (23Riddell D.R. Graham A. Owen J.S. J. Biol. Chem. 1997; 272: 89-95Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), supposedly mediated by ApoER2 (24Riddell D.R. Vinogradov D.V. Stannard A.K. Tagalakis A.D. Chadwick N. Sun X.M. Sutar A.K. Owen J.S. Atherosclerosis. 1999; 144 (Suppl. 1): 83Abstract Full Text PDF Google Scholar, 25Riddell D.R. Vinogradov D.V. Stannard A.K. Chadwick N. Owen J.S. J. Lipid Res. 1999; 40: 1925-1930Abstract Full Text Full Text PDF PubMed Google Scholar). Furthermore, the stimulation of androgen synthesis in rat ovarian theca cells by apoE also seems to be mediated by members of the LDLR family (26Zerbinatti C.V. Dyer C.A. Biol. Reprod. 1999; 61: 665-672Crossref PubMed Scopus (18) Google Scholar). A key feature of development of atherosclerosis is the activation of endothelial cells (27Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9959) Google Scholar). Evidence is accumulating to indicate that activation of the transcription factor activator protein-1 via the c-Jun NH2-terminal kinase (JNK) pathway plays a pivotal role in endothelial cell activation by cytokines, hypoxia, shear stress, and most interestingly, by LDL (28Zhu Y. Liao H.L. Wang N. Friedli Jr., O. Verna L. Stemerman M.B. Biochim. Biophys. Acta. 1999; 1436: 557-564Crossref PubMed Scopus (20) Google Scholar, 29Zhu Y. Lin J.H. Liao H.L. Friedli Jr., O. Verna L. Marten N.W. Straus D.S. Stemerman M.B. Arterioscler Thromb. Vasc. Biol. 1998; 18: 473-480Crossref PubMed Scopus (73) Google Scholar, 30Wang N. Verna L. Hardy S. Forsayeth J. Zhu Y. Stemerman M.B. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2078-2084Crossref PubMed Scopus (68) Google Scholar). The JNK-signaling pathway is involved in a variety of processes including cellular responses to environmental stress, cell proliferation, apoptosis, and morphogenesis (for review see Ref. 31Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1377) Google Scholar). Recently it became evident that this pathway plays an important role in regulating region-specific apoptosis of neurons during early brain development (32Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5028) Google Scholar, 33Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar). The purpose of the current study was to search for proteins interacting with the proline-rich insertion in the cytoplasmic tail of ApoER2 and to examine if this region is involved in signaling pathways. Here we demonstrate that this domain interacts with the JNK-interacting protein (JIP)-1 and JIP-2, both members of the JIP group of mitogen-activated protein kinase scaffolding proteins (34Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (406) Google Scholar). Furthermore, we show that the occurrence of the proline-rich domain-containing splice variant of ApoER2 and the expression of JIP-2 coincide during differentiation of neurons. The results demonstrate a molecular link between ApoER2 and the JNK signaling pathway. The cDNA coding for the 59-amino acid insertion of the murine ApoER2 tail was generated using the following primers: sense, 5′-GGA ATT CGC AAT CAG CAA CTA TGA TCG C and antisense, 5′-TTG GAT CCT TAC TTG CAC TTG ACG ACA GGC. The PCR product was cloned via internal EcoRI restriction sites of the primers into pLexA. Fidelity of the construct was confirmed by sequencing, and the construct was subsequently used as a bait for screening a mouse brain MatchmakerR LexA Yeast Two Hybrid library (CLONTECH) according to the manufacturer's instructions. Putative positive colonies were streaked out to create master plates, and the corresponding plasmid insertions were selectively amplified by colony PCR using vector-specific primers and were directly sequenced. The full-length cDNA of murine JIP-2 was cloned by two consecutive rounds of 5′-rapid amplification of cDNA ends PCR. Using primer a (5′-ACTGGCCTCGCAGCTGCTGTCC, annealing temperature, 58 °C), a 1678-base pair fragment was obtained. Then, using primer b (5′-GTCGTCCGTGATCTCAGACAGG, annealing temperature, 58 °C), another 81 base pairs of the 5′-sequence were obtained. The ATG assigned to represent the start codon for the mouse JIP-2 cDNA is located within a sequence consistent with the consensus sequence of translational start sites (35Kozak M. Nucleic Acids Res. 1987; 15: 8125-8132Crossref PubMed Scopus (4161) Google Scholar) and corresponds to the published sequence of human JIP-2 (34Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (406) Google Scholar). An IB1 (JIP1-b) cDNA was obtained from Dr. G. Waeber, Lausanne, France. IB1 and JIP-2 were tagged at the 5′-end with a 9xmyc epitope and cloned into pCIneo (Promega). The partial cDNA clone obtained by Yeast Two Hybrid screening (corresponding to amino acids 607–830, Fig.3 B) was tagged at the 5′-end with an influenza hemagglutinin (HA) epitope and cloned into pCIneo. GST fusion proteins of the respective domains of the ApoER2 tail (Fig. 1) were prepared as described (9Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) using the following primers: A) sense, 5′-CGG GAT CCG CCG GAA GAA CAC CAA GAG C; antisense, 5′-GGA ATT CTC AGG GCA GTC CAT CAT C (tail with and without proline-rich insertion; the resulting two bands were separately subcloned and verified by sequencing); and B) sense, 5′-CGG GAT CCG AGC AAT CAG CAA CTA TGA TC; antisense, 5′-GGA ATT CTC ACT TGC ACT TGA CGA C (proline-rich insert). GST-Dab1 is described in Ref. 14Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar; full-length Dab1 was tagged at the 5′-end with the HA epitope and cloned into pCI neo. The cDNA constructs corresponding to the SH3 (270 base pairs) and the PID (430 base pairs) domains of JIP-2 were amplified via PCR from the full-length JIP-2 cDNA using the following primers: SH3 sense, 5′-GAATTCAATGTCAACAGCACGTCTCGATCC; SH3 antisense, 5′-CCAGCAGGTCCTTGGCAGGACC (annealing temperature 60 °C); PID sense, 5′-GAATTCAAGGTGGATCGCTTCGATGTGC, PID antisense, 5′-GTAGATATCCTCTGTAGGGCAGGC (annealing temperature 60 °C). The PCR fragments were subcloned into a T/A cloning vector (pCR2.1, Invitrogen), then cut out via EcoRI sites present in the primers, and cloned into the mammalian expression vector pCIneo, which contained a 9xmyc-tag 5′ of the insertion. Fidelity of all constructs was verified by sequencing.Figure 1Structural organization of murine ApoER2 and of respective constructs used in the two hybrid screen and subsequent pull-down assays. a, cartoon of full-length ApoER2 emphasizing relevant structural features of the receptor. (open squares, ligand binding repeats; the vertical double line indicates plasma membrane; hatched box, intracellular domain without proline-rich region; open box, proline-rich insertion). The sequence of the proline-rich insertion is presented in detail above the receptor. b, lex-A fusion construct corresponding to the proline-rich insertion used for the two hybrid screen. c and d, GST-fusion constructs corresponding to the respective tail variants used for pull-down experiments presented in Figs. 2 and 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Total RNA was prepared from different tissues of a male and female Balb/C mouse using TRI REAGENT (Molecular Research Center, Inc.). 30 μg of total RNA was separated by electrophoresis on a 1.5% agarose gel and transferred onto a nylon membrane. After UV cross-linking, hybridization was performed using the partial JIP-2 cDNA originally found in the Yeast Two Hybrid screen. Methylene Blue staining was used as quality and loading control. The antibody against mouse ApoER2 was produced against the complete intracellular domain expressed as a GST fusion protein as described previously (9Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Anti-JIP-2 was produced against a synthetic peptide (see Fig. 3 A) corresponding to the cloned mouse JIP-2 cDNA as described (4Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The following antibodies were obtained commercially from the sources indicated: antimitogen-activated protein 2 (Sigma), anti-HA tag (16B12, Babco), anti-Myc (9E10, used as hybridoma supernatant from the corresponding cells, ATCC), Oregon Green 488-labeled goat anti-mouse IgG (Molecular Probes), Alexa 594-labeled goat anti-rabbit (Molecular Probes), and goat anti-rabbit-biotinylated IgG (Sigma). 293 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and 584 mg/liter glutamine and transfected with Lipofectin reagent (Life Technologies, Inc.). Cellular extracts for Western blots and precipitation experiments were prepared by lysing the cells in HUNT buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mmEDTA, 0.5% Nonidet P-40, 0.1 mg/ml phenylmethylsulfonyl fluoride) for 15 min on ice and centrifugation to remove insoluble material. For pull-down experiments, 30 μl of cell extract were diluted with 150 μl of HUNT buffer. After the addition of ∼10 μl of glutathione-Sepharose with bound fusion protein, the mixture was incubated for 2 h at 4 °C on a rotary shaker. Subsequently, the beads were washed three times with 200 μl of HUNT buffer. Bound proteins were eluted by incubating the beads for 3 min at 95 °C under reducing conditions in SDS loading buffer and subjected to SDS-polyacrylamide gel electrophoresis. Electrophoresis, transfer to nitrocellulose membranes, and Western blotting were performed as described previously (36Hiesberger T. Hermann M. Jacobsen L. Novak S. Hodits R.A. Bujo H. Meilinger M. Hüttinger M. Schneider W.J. Nimpf J. J. Biol. Chem. 1995; 270: 18219-18226Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Mice were euthanized with CO2 and perfused via the left ventricle with 25 ml of PBS, followed by 25 ml of a solution containing 75 mml-lysine, 75 mm sodium phosphate, pH 7.3, 2% paraformaldehyde, and 2.4 mg/ml sodium meta-periodate. The brain, testis, and epididymus were then removed and stored in the fixation solution for 4 h at 4 °C. Embedding and specimen preparation was done as described (9Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Incubation with the primary antibodies (dilutions: anti-JIP-2, 1:500; anti-ApoER2, 1:100) was performed at 4 °C for 20 h in blocking solution. After five washes in PBS, the following incubations were performed at 23 °C: goat anti-rabbit biotinylated IgG diluted 1:500 in blocking solution for 1 h, five washes with PBS, peroxidase-labeled avidin (Sigma) for 1 h diluted 1:200 in 1% nonfat dry milk in PBS, and a final wash with PBS (five times). For the color reaction, slices were incubated in 0.1 m sodium acetate, pH 5.1, containing 150 μl of 30% peroxide and 20 mg of 3-amino-9-ethylcarbazole, per 100 ml of buffer. The staining process was followed under the microscope (Zeiss Axiovert 135) and stopped by immersing the slides in water. Nuclei were counterstained with Harris-modified hematoxylin solution (Sigma). Neuronal stem cell culture and differentiation into neurons was performed according to Ref. 37Johe K.K. Hazel T.G. Muller T. Dugich-Djordjevic M.M. McKay R.D. Genes Dev. 1996; 10: 3129-3140Crossref PubMed Scopus (1035) Google Scholar. Embryos 15–17 days old were removed and put into PBS plus chlortetracycline (Life Technologies, Inc.). Embryo heads were opened sagittally under sterile conditions, and the brains were removed with forceps and transferred into HBSS (Life Technologies, Inc.). After three washes with HBSS, brains were triturated in Hanks' balanced salt solution and centrifuged at 4000 rpm for 4 min, and the cells were suspended in prewarmed culture medium (Dulbecco's Nut Mix F12 (Life Technologies, Inc.) supplemented with 2 mml-glutamine, B27 supplement (Life Technologies, Inc.), antibiotic/antimycotic (Life Technologies, Inc.)), grown on 10-cm cell culture dish coated with poly-l-ornithine (Sigma) at 37 °C with 5% CO2. Stem cells were grown in the presence of 10 ng/ml human recombinant bovine fibroblast growth factor (Sigma). Growth factor was added every day, and the medium was changed every second day. To start differentiation into neurons, in addition to 10 ng/ml basic fibroblast growth factor, 10 ng/ml recombinant platelet-derived growth factor-BB (Sigma) was added to the medium. Two days later only platelet-derived growth factor-BB was added daily. At 50% confluency, cells were split by rinsing once with PBS and incubating with 1 ml of Trypsin (Life Technologies, Inc.) for 10–20 min at 37 °C until cells detached. Trypsinization was stopped by the addition of medium supplemented with 1 mg/ml chicken egg white trypsin-inhibitor (Sigma). Poly(A)+ RNA from stem cells and differentiated neurons was prepared from five 10-cm cell culture dishes each using the Micro FastTrackTM 2.0 mRNA isolation kit (Invitrogen). The resulting mRNA pellet was directly used for cDNA synthesis using oligo(dT) primers and SuperScript reverse transcriptase (Life Technologies, Inc.). The following primers were used: ApoER2, sense, 5′-CAGTGGCTGTCCCTCACTCGG and antisense, 5′-GAAGATGATGGACTGCCCTG (annealing temperature, 62 °C); IB1 (JIP-1b), sense, 5′-ATGTCTTCATGAGTGGCCG and antisense, 5′-GCTGTGTCCTTGAGATCAGTG (annealing temperature, 56 °C); JIP-2, sense, 5′-ACCTTGAGATCTCTCTGCGG and antisense, 5′-GGATATCTACCTGGAGTAAGC (annealing temperature, 60 °C). The PCR products were analyzed by agarose gel electrophoresis on 2% agarose. 293 cells were grown on culture slides (Becton Dickinson) coated with poly-d-lysine to a confluency of 50–70%. Mouse brain cells were transferred to coated 8-well slides 2–6 days prior to the staining. Slides were coated with poly-l-ornithine (Sigma) and fibronectin (Sigma). After washing twice with PBS, cells were fixed with a mixture of equal amounts of methanol and acetone at −20 °C for 4 min (293 cells) or at −20 °C for 30–60 min (mouse brain cells). Fixed cells were rehydrated for 15 min at room temperature with PBS, incubated with the primary antibody in blocking solution (1% nonfat dry milk in PBS) for 1 h at room temperature, washed three times with blocking solution, and incubated at room temperature with the secondary antibody (Alexa 594 1:500 or Oregon Green 488 1:500, Molecular Probes) for 1 h. After two washes with blocking solution and two final washes with PBS, microscopy was performed. The intracellular domain of ApoER2 is highly homologous to that of LDLR and VLDLR and binds the intracellular adaptor protein disabled via its conserved NPXY motif (11Trommsdorff M. Borg J.-P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33565Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). However, because of the differential splicing of an additional exon in the ApoER2 gene, a region coding for a 59-amino acid insertion is present in the cytoplasmic tail of a significant fraction of the receptor population (7Brandes C. Novak S. Stockinger W. Herz J. Schneider W.J. Nimpf J. Genomics. 1997; 42: 185-191Crossref PubMed Scopus (50) Google Scholar). As depicted in Fig. 1 a, this proline-rich domain is inserted 10 amino acids before the carboxyl terminus of the protein. To identify proteins interacting with this specific domain of ApoER2, we used the yeast two-hybrid method based upon the LexA system (38Mendelsohn A.R. Brent R. Curr. Opin. Biotechnol. 1994; 5: 482-486Crossref PubMed Scopus (54) Google Scholar). A Matchmaker LexA mouse brain cDNA library was screened using the 59-amino acid insert as bait (Fig.1 b). Out of 108 transformants screened, four clones scored positive for an interaction of the receptor insert with products of transcripts encoded by the library. Two of these clones represented sequences interrupted by stop codons in all reading frames; the remaining two clones were identical and homologous to human JIP-1 and JIP-2 (34Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (406) Google Scholar). To verify the interaction between ApoER2 and the candidate protein at the biochemical level, we performed “pull-down” experiments by incubation of extracts prepared from 293 cells expressing the HA-tagged protein with bacterially expressed fusion proteins between GST and various receptor tail constructs as shown in Fig. 1, c–e. Protein complexes were precipitated with glutathione-Sepharose beads, eluted, separated on SDS-polyacrylamide gel electrophoresis, and subjected to Western blotting with a monoclonal anti-HA antibody (Fig.2, lanes 2–4). The same antibody was used to demonstrate the expression of the tagged cDNA product in transfected 293 cells by direct Western blotting of the respective cell extracts (Fig. 2, lane 1). Clearly, the HA-tagged product of the candidate clone interacted with the GST fusion proteins that contained either the proline-rich insert alone (“i,”lane 2) or the full-length tail (“ti,” lane 3) of the receptor. Importantly, the interaction is specific for the insert, because the tail without insert (“t,” lane 4) did not interact with the HA-tagged protein. The specificity for the insert is also documented by the fact that the tagged protein does not bind to the cytosolic tail of the VLDLR (data not shown). To further verify this result, we used an HA-tagged fragment of Dab1, which previously had been shown to bind to the ApoER2 tail independently of the proline-rich insert (14Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar), in a similar experiment. Indeed, Dab1 binds to both the short and long cytosolic domains but not to the proline-rich insert itself (Fig. 2, lanes 5–8). In contrast to the novel protein identified by the screen, it was previously shown that Dab1 binds to the VLDLR tail, too (14Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar). Having biochemically verified the interaction of the product of the candidate cDNA obtained in the tw" @default.
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• W2022840078 date "2000-08-01" @default.
• W2022840078 modified "2023-10-18" @default.
• W2022840078 title "The Reelin Receptor ApoER2 Recruits JNK-interacting Proteins-1 and -2" @default.
• W2022840078 cites W1500815560 @default.
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